1. Field of the Invention
This invention relates generally to antibody purification. In particular, the invention relates to a method for recovering an antibody fragment from variants, impurities, and contaminants associated therewith.
2. Description of Related Art
Hydrophobic interaction chromatography (HIC) is a useful tool for separating molecules based on their hydrophobicity. Generally, sample molecules in a high salt buffer are loaded on the HIC column. The salt in the buffer interacts with water molecules to reduce the salvation of the molecules in solution, thereby exposing hydrophobic regions in the sample molecules which are consequently adsorbed by the HIC column. The more hydrophobic the molecule, the less salt needed to promote binding. Usually, a decreasing salt gradient is used to elute samples from the column. As the ionic strength decreases, the exposure of the hydrophilic regions of the molecules increases and molecules elute from the column in order of increasing hydrophobicity. Sample elution may also be achieved by the addition of mild organic modifiers or detergents to the elution buffer. HIC is reviewed in Protein Purification, 2d Ed., Springer-Verlag, New York, pgs 176-179 (1988).
HIC has been used by various researchers for purification of antibodies. Danielsson et al., Journal of Immunological Methods 115:79-88 (1988) found that HIC was particularly useful for purification of monoclonal antibodies from mouse ascites when the isoelectric point of the antibodies was below 7.2. HIC was performed with an Alkyl SUPEROSE HR™ column. The buffer system was 0.1 M phosphate, with addition of ammonium sulfate. Usually the starting buffer contained 2M ammonium sulfate. Bridonneau et al., Journal of Chromatography 616:197-204 (1993) were interested in determining whether or not different HIC columns could be used for selective purification of human immunoglobulin G (IgG) subclasses. The antibodies were adsorbed on Phenyl-, Butyl-, or Octyl-SEPHAROSE™ columns in 1M ammonium sulfate (pH 7.0) and eluted with decreasing salt gradient. Octyl-SEPHAROSE™ medium yielded a poorly adsorbed fraction somewhat enriched in IgG2a. See also Berkowitz et al., Journal of Chromatography 389:317-321 (1987); Gagnon et al. (90th Annual Meeting, American Society for Microbiology, Anaheim, May 13-17, 1990) Abstract No. 0-4; Johansson et al. Biol. Recombinant Microorg. Anim. Cells, (Oholo 34 Meeting), 409-414 (1991); Pavlu et al., Journal of Chromatography 359:449-460 (1986) and Abe et al., Journal of Biochemical and Biophysical Methods 27:215-227 (1993) concerning HIC of antibodies.
HIC has also been used for purifying antibody fragments. Inouye et al., Protein Engineering, 6(8):1018-1019 (1993); Inouye et al., Animal Cell Technology: Basic & Applied Aspects 5:609-616 (1993); Inouye et al., Journal of Biochemical and Biophysical Methods 26:27-39 (1993) and Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) prepared F(ab′)2 fragments from pepsin digests of mouse IgM monoclonal antibodies using a TSKgel Ether-5PW™ HIC column. The antibody fragments were salted out with 60% ammonium sulfate and the precipitates were dissolved into phosphate-buffered saline (PBS, pH7.4) containing 1 M ammonium sulfate. This solution was loaded onto the HIC column which had been equilibrated with PBS also containing 1M ammonium sulfate. The F(ab′)2 fragments which were adsorbed onto the column were eluted by reducing the ammonium sulfate concentration in the elution buffer to 0M. Inouye et al. found that the fraction containing the F(ab′)2 was homogeneous by both SDS-PAGE and gel filtration HPLC. The method was considered to be suitable for large-scale purification of F(ab′)2 fragments. Similarly, Rea et al., Journal of Cell. Biochem. Suppl. 0, Abstract No. X1-206 (17 Part A), p. 50 (1993) evaluated HIC for purification of a F(ab′)2 fragment produced by peptic digestion of a murine IgG2a monoclonal antibody. Protein A purification for removal of residual intact antibody preceded the HIC step. The purification performance of three different HIC columns was tested at several different salts and pHs. POROS PE™ (Phenyl ether) was found to be the best column and phosphate-buffered sodium sulfate at pH 8 gave the best resolution of the F(ab′)2 fragment.